A role and mechanism for redox sensing by SENP1 in β-cell responses to high fat feeding

Pancreatic β-cells respond to metabolic stress by upregulating insulin secretion, however the underlying mechanisms remain unclear. Here we show, in β-cells from overweight humans without diabetes and mice fed a high-fat diet for 2 days, insulin exocytosis and secretion are enhanced without increased Ca2+ influx. RNA-seq of sorted β-cells suggests altered metabolic pathways early following high fat diet, where we find increased basal oxygen consumption and proton leak, but a more reduced cytosolic redox state. Increased β-cell exocytosis after 2-day high fat diet is dependent on this reduced intracellular redox state and requires the sentrin-specific SUMO-protease-1. Mice with either pancreas- or β-cell-specific deletion of this fail to up-regulate exocytosis and become rapidly glucose intolerant after 2-day high fat diet. Mechanistically, redox-sensing by the SUMO-protease requires a thiol group at C535 which together with Zn+-binding suppresses basal protease activity and unrestrained β-cell exocytosis, and increases enzyme sensitivity to regulation by redox signals.

to be straightened out.I am most concerned about how some of the data is handled in the statistical analyses.have listed some comments to the authors below 1. Please provide a table summarizing the characteristics of the human donors 2. The statistical tests used are indicated in each figure legend, however, it is unclear to the reader which statistical tests have been used for which figure panel.1B: it is unclear to me if the number of cells (63-236 and 21-36) is cells per donor or total number of cells analysed for non-diabetic donors and donors with T2D, respectively.

Figure
And a "follow-up" question, is the statistical analysis performed on the donor data (i.e.11-33 vs 4-5), or cells (i.e.63-236 vs 21-36)? 4. Figure 1A and C: what is the "control" in the legend on the y-axis? 5. Figure 1B: please indicate in the results paragraph or figure legend that this is patch clamp experiments, otherwise the reader (at least I did until I came to the methods) will wonder if the cells are sorted and/how they were identified as β-cells.
6.If I understand correctly, figure 1A and 1B shows β-cell secretion at different glucose levels, one in the form of actual secretion (measurment of releases insulin), and the other in the form of exocytosis (patch clamp measurements).However, I do not understand why you have significant differences between BMI <25 ND and BMI >25 ND at 10mM glucose in A but not in B (and vice versa at 1mM) but I am guessing this is due to the nature of the experiments.Regarding the significant difference at 1mM glucose in 1B you write "suggesting an increased insulin granule priming or amplifying effect even at low glucose".Would this not be detected also in the experiments in Fig 1A , and if there is priming/amplifying effects, would these not also affect secretion at high glucose?Please discuss/clarify.7. Figure 1A-D: Please discuss potential reasons for why the data looks different in older humans 8. Figure 1H and supp figure 1C: the insulin content is expressed as a concentration (pg/ml), please convert this to an amount (pg/islet for example).Also, what are the dots representing?The number of dots does not seem to match the numbers in the respective legend 9. Figure S1D-E 2A: you write "n=216 genes, N=4, 3 mice", does this mean that islets from 4 CD and 3 HFD mice were analysed?These are very small numbers and ideally should be increased.
12.There is no reference to the supplementary table in the paragraph on RNA seq, please add.And the table needs more information, at least mean expression and SD for both groups.
13.If I understand the methods section correctly you consider genes with p<0.1 significant in the RNAseq data?And there is no correction for multiple testing.I feel this is not stringent enough, please provide a reasoning behind this or use stricter criteria.The p-value for your main candidate Senp1 is an uninpressive 0.092.A larger number of animals in this analysis (see comment 12) will help.
14. Figure 2B: please provide the full list of significant pathways in a supp table, including information on which genes in these pathways that are differentially expressed.
15.Why did you use two different methods to measure oxygen consumption and way are they giving different results)?I.e., why may it be significant at 2.8mM glucose in B but not in A?
16.The oligomycin-sensitive respiration (i.e. the drop after addition of oligomycin) and maximum respiration looks quite different between the 2 groups.Can you do similar graphs as in 3B for those?Or specify in the text that there are not significant differences for these parameters?And irrespective of what you chose to do, please present the p-values for those comparisons in the figure or text.17.Can you indicate the different respiratory parameters you measure in Figure 3A?For example, you write that respiration driving proton leak is higher in the 2-day HFD islets and those not familiar with mitochondrial respiration will probably believe that proton leak is simply the values that the data points indicate in the graph while in realty it's the difference between the values at the data points and the non-mitochondrial respiration.
18. Figure 3C: if I understand correctly, you have analysed these data as if you have n=65+41.This is not correct, you have n=6+6 (this is the same issue I tried to raise in comment 2 above, if my point was unclear).
21. Figure 3G: you write N=2-9 mice, but there is no bar with less than 4 dots, and several bars have more than 9 dots.What do the dots represent?22. Figure 3H: same as comment 14 and 15.This is true for several other figure panels both in the main file and in the supplementary.I will stop commenting on it, but in my view this way of analysing the data is not ok and needs to be changed throughout the manuscript.
23. Figure 4E-F: there is no indication of significance for CD WT mice at 2.8 mM compared to 10mM.Was it not significant?24.For the GTTs you sometimes use 1g/kg and sometimes 0.5g/kg.Why? 25. Figure 5D: you write that infusion of WT SENP1 increased exocytosis but there is no indication of significance in the graph, please add (or are the 2 stars the significance of that comparison?If so, clarify).
26.In the discussion there are references to data not mentioned in the results section.
Maybe Nat Com has other rules regarding this, but if you ask me all data mentioned in the discussion should be mentioned in the results section.
27.In the methods you mention ITTs, but there is no ITT data in the manuscript 28.In the methods section on in vitro Senp1 assays you refer to Fig. S2A which is an IPGTT figure.

Reviewer #3 (Remarks to the Author):
The authors have explored molecular mechanisms affecting pancreatic beta-cell insulin secretion in response to metabolic stress.Using in vitro cell based assays, they demonstrate that insulin secretion and exocytosis is enhanced in beta-cells from overweight humans and mice fed a 2-day high-fat diet without increased calcium influx.RNA-Seq analysis of mouse beta-cells isolated following a 2-day high fat diet revealed early changes in metabolic pathways and increases expression of the SENP1 SUMO protease.Enhanced insulin secretion and exocytosis following a 2-day high-fat diet was found to be dependent reduced intracellular redox.In addition, the SENP1 SUMO protease was found to be required using SENP1 pancreas and beta-cell knockout mice.In vitro assays using recombinant SENP1 catalytic domain revealed that redox sensitivity was dependent on C535 and modulated by Zn+.Cell-based assays supported roles for C535 and Zn+ in affecting SENP1 activity and its role in affecting beta-cell exocytosis and insulin secretion.
Previous studies by this group and others have supported roles for the SENP1 SUMO protease in regulating insulin-secretion by beta-cells.Previous studies have also indicated that SENP1 activity is subject to redox regulation.The specific molecular mechanisms by which SENP1 controls beta-cell function and how this may be impacted by changes in redox potential of the cells, however, has remained uncertain.The studies reported in this manuscript address these important questions and provide new and potentially valuable insights.
Overall, the manuscript was a challenge to read and understand.This could be addressed by more clearly explaining specific questions underlying individual experiments and putting the results in the context of these questions.Moreover, more background information and context is needed to appreciate the significance of experiments looking at exocytosis and insulin secretion.Are the experiments particularly novel and significant because of the short, 2-day, high-fat diet?Or because the experiments are looking specifically at exocytosis?For someone not in the diabetes field, it is unclear how novel these experiments are.Have these specific questions not already been addressed in other studies?What makes the specifics of the RNA-Seq experiment novel?
A second major concern is the complete absence of any discussion of sumoylation.It is unclear whether the authors think that SENP1 is functioning as a SUMO protease to affect beta-cell function, or in some other capacity.Some discussion is needed.Experiments evaluating effects of SENP1 on overall sumoylation levels in experiments involving introduction of recombinant proteins would also be very informative.Do effects or lack of effects of SENP1 on exocytic capacity and insulin secretion correlate with effects on sumoylation?
More specific comments are as follows: (1) In Figure 1A, insulin release is similar in all three groups tested at 1mm glucose.In Figure 1B, exocytic capacity is shown to be higher in the BMI > 25 ND group at 1 mm glucose.Why do exocytic capacity and glucose release not correlated at 1 mm glucose?
(2) For many experiments (1A-D, 4E and F…), there is no data collected at 0 glucose.In 4E and F this is a concern because it is concluded that loss of SENP1 prevents up-regulation of exocytosis.Without a baseline of exocytosis in the absence of glucose, it is unclear if there is no up-regulation, or if the level of upregulation is decreased.
(3) Intracellular dialysis or infusion is used to introduce recombinant SENP1 proteins into cells in multiple experiments.The authors should provide and explanation of this technique and details of SENP1 expression.What is the efficiency of the technique in terms of amount of protein delivered, and fraction of cells taking up the protein?In addition, it would be helpful to have information of the intracellular localization of the SENP1 protein.The Nterminus of SENP1 is known to contain determinants of nuclear localization.Where does the catalytic domain concentrate in the beta-cells?Also, as discussed above, possible effects on introducing recombinant proteins into cells on sumoylation levels would be informative.
(4) What is GRX1 and why does it influence the effect of GSH on SENP1 activity (Figure 5E)?
(5) In Figure 5D, the authors switch to human beta-cells.It would be more informative to see this experiment done in beta-cells isolated from WT and betaSENP1 KO cells.(6) The results of Figure 5B and 5C are confusing.First, the authors show that the 535S mutant is 2-fold more active and then they show that it has the same activity.There is some mention that "some recombinant proteins were likely partially inactivated by oxidation".This section needs to be cleaned using proteins of similar quality.
(7) The proton transfer pathway (Figure 6SB) to explain SENP1 redox-sensing is intriguing, but needs additional support.In particular, do mutations of H533 or D550 support this model?
: you write 8+8 and 7+7 mice, but there are many more dots in the graphs.